![Figure 5. CD40LG-edited X-HIGM T cells restore in vitro naive B-cell CSR. (A) Induction of CSR in healthy donor naive B cells. (Left panel) Naive B cells incubated in the presence of IL-21 and CPG ODN (CSR Mix) and α-IgM, with or without sCD40L. Low levels of background CSR (IgG+ B cells) were observed in the absence of sCD40L (3.5%, top); addition of sCD40L increased CSR (10%, bottom). (Right panel) Allogeneic naive IgG− B cells cocultured with CSR Mix and resting (top panel) or sCD40L-activated (bottom panel) T cells. Representative plots of CD19+ B-cell population after coculture with mock-edited or sorted edited healthy donor T cells are shown. Activated edited cells induced CSR at comparable levels to mock-edited or sCD40L-treated conditions. (B) Average percentage of class-switched B cells (IgG+) from 3 independent experiments (each using T cells from different healthy donors) demonstrate nonsignificant differences in class-switching rates vs activated nonedited (mean ± standard error of the mean [SEM] = 9.6 ± 3.9) and activated edited cells (CD40LG[GFP.CD40L.3′UTR], 5.33 ± 3.19; CD40LG[GFP.CD40L.WPRE], 9.42 ± 1.85; P = .16 and P = .92, respectively). (C) IgG CSR of allogeneic (healthy donor) naive B cells in coculture with edited or mock-edited X-HIGM T cells. Representative data set of B-cell (CD19+) CSR using 2 X-HIGM T-cell donors (row 1 and row 2) which were either mock-edited (left column), CD40LG[GFP.CD40L.3′UTR] edited (middle column), or CD40LG[GFP.CD40L.WPRE] edited. (D) Bar graph summarizing CSR (percentage IgG+) normalized to background from 9 independent experiments (each in duplicate or triplicate) using 4 X-HIGM donors. Bars represent average fold-increase of naive B-cell IgG CSR induced by activated X-HIGM cells, compared with that of resting mock-edited X-HIGM T cell (background). Average ± SEM: Mock = 1.92 ± 0.27; CD40LG[GFP.CD40L.3′UTR] = 4.37 ± 0.96; CD40LG[GFP.CD40L.WPRE] = 5.64 ± 1.32. Editing of X-HIGM T cells induced a significant increase in B-cell CSR compared with nonedited X-HIGM cells (P = .0081 and P = .0084, respectively).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/127/21/10.1182_blood-2015-11-683235/4/2513f5.jpeg?Expires=1722710491&Signature=dQVz5rR4Da~amUPPryR49le-6qGRdNIaAkX-R~TaUSupLZjDmQ8qnidumn8pFGCdFSw-v78uVffGw9~Qtsm0cP9Q7AeNVnwMLmTgddrj70BYb28PYvKUAlOfQ7aNzsRAcO~Djs8b7TYn4u1LqUcLXIF0Or~7mDer-rBSyLukrZnHtBmFHE8DL-GPr6GzSykF0dfutF1jrEx7XHmtklIaUaNqAsIrMrPjLSPA1TkfmOQcQkDobViLJj626NRvvN-rpTUoTumMEbnmuwpG7EIHQddSifMrNZ21RidYEcx7EaOxcRXHm~D9sqIfAmbhvdjgtKmgZ9y0LVZTXGg3v341EQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD40LG-edited X-HIGM T cells restore in vitro naive B-cell CSR. (A) Induction of CSR in healthy donor naive B cells. (Left panel) Naive B cells incubated in the presence of IL-21 and CPG ODN (CSR Mix) and α-IgM, with or without sCD40L. Low levels of background CSR (IgG+ B cells) were observed in the absence of sCD40L (3.5%, top); addition of sCD40L increased CSR (10%, bottom). (Right panel) Allogeneic naive IgG− B cells cocultured with CSR Mix and resting (top panel) or sCD40L-activated (bottom panel) T cells. Representative plots of CD19+ B-cell population after coculture with mock-edited or sorted edited healthy donor T cells are shown. Activated edited cells induced CSR at comparable levels to mock-edited or sCD40L-treated conditions. (B) Average percentage of class-switched B cells (IgG+) from 3 independent experiments (each using T cells from different healthy donors) demonstrate nonsignificant differences in class-switching rates vs activated nonedited (mean ± standard error of the mean [SEM] = 9.6 ± 3.9) and activated edited cells (CD40LG[GFP.CD40L.3′UTR], 5.33 ± 3.19; CD40LG[GFP.CD40L.WPRE], 9.42 ± 1.85; P = .16 and P = .92, respectively). (C) IgG CSR of allogeneic (healthy donor) naive B cells in coculture with edited or mock-edited X-HIGM T cells. Representative data set of B-cell (CD19+) CSR using 2 X-HIGM T-cell donors (row 1 and row 2) which were either mock-edited (left column), CD40LG[GFP.CD40L.3′UTR] edited (middle column), or CD40LG[GFP.CD40L.WPRE] edited. (D) Bar graph summarizing CSR (percentage IgG+) normalized to background from 9 independent experiments (each in duplicate or triplicate) using 4 X-HIGM donors. Bars represent average fold-increase of naive B-cell IgG CSR induced by activated X-HIGM cells, compared with that of resting mock-edited X-HIGM T cell (background). Average ± SEM: Mock = 1.92 ± 0.27; CD40LG[GFP.CD40L.3′UTR] = 4.37 ± 0.96; CD40LG[GFP.CD40L.WPRE] = 5.64 ± 1.32. Editing of X-HIGM T cells induced a significant increase in B-cell CSR compared with nonedited X-HIGM cells (P = .0081 and P = .0084, respectively).
